It has been increasingly appreciated that perturbations in epigenetic processes are associated with diverse human diseases such as cancer and immunodeficiency disorders. To date, more than 100 histone protein posttranslational modifications (PTMs) have been described. However, characterization of the enzymes that catalyze the modifications and the proteins that recognize and transduce specific PTMs to biological outcomes remains largely underdeveloped. Given the association of aberrant PTMs with human disease, there is a compelling need to develop innovative tools to characterize proteins involved in epigenetic regulation. We propose to develop a new technology utilizing protein arrays for high-throughput coverage of the human epigenome. The overall goals are to establish methods for protein array technology to be applied for epigenetic research, and validate protein array technology as a new tool in the discovery of how chromatin modification events regulate epigenetic biology. In this proposal, we will focus our efforts on developing a random fixed methyl-lysine Oriented Peptide Array Library (OPAL) which will allow the rapid screening of all methyl-lysine binding proteins (effectors) and lysine methyltransferases (enzymes) for their substrate or binding specificities. In order to interrogate all effectors and enzymes, we will synthesize four oriented KGXXXKXXXGK-biotin peptide libraries [X represents random amino acids (any amino acid except cysteine)] in which the (1) central- K is unmethylated; (2) central- K is mono-methylated; (3) central- K is di-methylated; and (4) central- K is tri-methylated. The OPAL platform will be validated by determining the binding specificities of known methyl-lysine effector proteins (HP1, RAG2, ORC1, and PHF19). In addition, we will use this platform to confirm the established consensus substrates recognition sequences for the methyltransferases G9a and SET7/9. Success will be achieved and progression to Phase 2 justified if we observe that the consensus targets for the effectors (Aim 1) and enzymes (Aim 2) are consistent with published results. Given that these proteins will be screened against high complexity arrays the results of the OPAL screen may reveal novel targets for these effectors and enzymes, which will likely reveal new insights into the function of these factors since all lysine methylation states can be screened in a single experiment. In the long term, Epicypher will create a suite of OPAL platforms that include PTMs other than lysine methylation, such as lysine acetylation and arginine methylation, which will enable the company to serve as a valuable resource to the research community. More generally, it is becoming clear that lysine acetylation and methylation are not only found on histones, but on a large number of non-histone proteins as well. Thus, the proposed OPAL platforms would, for the first time, provide the resources necessary to define the function of orphan effectors and enzymes, and help the research community predict which non-histone proteins could be substrates and interacting partners for enzymes and effectors that have been traditionally associated with the histone code and serve as an important step in understanding the human lysine methylome.